A kind of fish scale composite hydrogel for drug loading and its preparation method

Abstract

The present invention discloses a fish scale composite hydrogel for drug loading and a preparation method thereof, wherein the method uses high-frequency and low-frequency sound waves to alternately treat fish scales, extracts fish scale collagen and hydroxyapatite by hot pressing and ionic liquid methods, respectively, and then cross-links acrylic acid-modified β-cyclodextrin with peptide calcium chelate to prepare the fish scale composite hydrogel. The present invention utilizes high-frequency and low-frequency sound waves to alternately treat fish scales, and synergistically dissociates the bonding structure of collagen and hydroxyapatite through cavitation effect and mechanical vibration, thereby improving the extraction efficiency while avoiding collagen denaturation; the prepared fish scale composite hydrogel has a "jellyfish" structure: a stable gel core layer structure is formed by a cross-linked network of hydroxyapatite and chitosan coated with β-cyclodextrin; a branched tentacle structure formed by covalently linking acrylic acid-modified β-cyclodextrin and collagen realizes drug gradient controlled release, and the strong adhesion of the tentacles can accurately locate the drug release site, providing innovative solutions for the fields of drug delivery and tissue engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to a fish scale composite hydrogel for drug loading and a preparation method thereof. Background Art

[0002] Fish scales are layered calcified tissues derived from the epidermis of fish, widely present in freshwater and marine fish processing by-products, and have the characteristics of natural renewability, rich resources, and low cost. Its multi-layer composite structure consists of a dense mineralized layer on the outer layer and a collagen fiber network on the inner layer, with both excellent mechanical strength and flexibility.

[0003] Fish scales are important biomineralized tissues on the body surface of fish, formed by the common secretion of the epidermis and dermis layers, and have a unique cross-scale composite structure. Fish scales mainly contain collagen and hydroxyapatite, and their structure has hydroxyapatite wafers as a rigid reinforcing phase, embedded in a flexible collagen fiber matrix to form an organic-inorganic hybrid plywood structure. Research shows that fish scales are generally irregular hexagons, and the surface is divided into a covering area and an exposed area, forming a microscopic corrugated topological morphology, which can effectively disperse external force impacts and protect the fish body from external mechanical damage.

[0004] The collagen content in fish scales can reach 20-30%, and it also contains active ingredients such as hydroxyapatite and glycosaminoglycans, endowing it with biocompatibility, degradability, and promoting cell adhesion and other properties. In recent years, with the development of the aquaculture processing industry, millions of tons of fish scale waste are generated globally every year. The traditional treatment methods mainly include landfill or incineration, which not only cause waste of resources but also may lead to environmental pollution. Therefore, developing high-value utilization technologies for fish scales, especially converting them into biomedical materials, has become a research hotspot in the fields of circular economy and green chemistry.

[0005] At present, the preparation methods of fish scale hydrogel mainly include acid / alkali extraction, enzymatic hydrolysis and physical modification. For example, patent CN201510855741.2 uses acid treatment-salting out-dialysis process to directly obtain collagen hydrogel. Although the steps are simplified, the strong acid environment easily destroys the tertiary structure of collagen, resulting in insufficient gel strength (<200g·mm). Enzymatic hydrolysis (such as trypsin hydrolysis) can improve the collagen extraction rate, but the enzyme cost is high and the product molecular weight distribution is wide, which requires ultrafiltration purification, increasing the complexity of the process. Although the physical method (such as high temperature boiling) is easy to operate, excessive thermal denaturation will reduce the biological activity of collagen, and the yield is limited by the water-scale ratio (the yield is only 3.62% at the optimal 3:1). In addition, the existing technology mostly relies on chemical cross-linking agents (such as glutaraldehyde), which may introduce cytotoxicity and limit its medical application. The existing patented technology generally ignores the reuse of hydroxyapatite inherent in fish scales, resulting in waste of mineral components and failure to achieve high value of all components. Therefore, these defects seriously restrict the industrial application of fish scale hydrogel in the fields of drug delivery, wound repair, bone regeneration, etc. Therefore, studying new preparation methods of fish scale hydrogel is conducive to the large-scale application of fish scale hydrogel in the fields of tissue engineering, drug delivery, etc., and promoting the resource utilization and high value transformation of aquatic waste. Summary of the invention

[0006] Technical problem to be solved: In view of the above technical problems, the purpose of the present invention is to disclose a fish scale composite hydrogel for drug loading and a preparation method thereof, which utilizes high-frequency and low-frequency sound waves to alternately treat fish scales to synergistically dissociate the bonding structure of collagen and hydroxyapatite through cavitation effect and mechanical vibration, thereby avoiding collagen denaturation and improving extraction efficiency; at the same time, the prepared fish scale composite hydrogel has a "jellyfish" structure: a stable gel core layer structure is formed by a β-cyclodextrin-coated hydroxyapatite-chitosan cross-linked network; a branched tentacle structure formed by covalently linking acrylic acid-modified β-cyclodextrin and collagen can achieve gradient controlled release of drugs, and the strong adhesion of the tentacles can accurately locate the drug release site, providing innovative solutions for the fields of drug delivery and tissue engineering.

[0007] Technical solution: A method for preparing a fish scale composite hydrogel, comprising the following steps:

[0008] S1. The fish scales are first cleaned, dried, crushed, and sieved to obtain fish scale powder, and water is added to prepare a fish scale powder solution;

[0009] S2. treating the fish scale powder solution with high-frequency-low-frequency sound waves, filtering the fish scale filtrate and the calcium ion-containing filtrate;

[0010] S3. The fish scale filtrate is added with water and then subjected to hot pressing to obtain fish scale collagen and residue, wherein the residue is subjected to ionic liquid extraction, dried to obtain hydroxyapatite, and a portion of the fish scale collagen is enzymatically hydrolyzed to obtain fish scale collagen peptides;

[0011] S4. Treat the fish scale collagen peptide and the calcium ion-containing filtrate obtained in S2 at pH 6.5 - 8.0 and 45 - 55 °C for 30 - 45 min to obtain a peptide-calcium chelate;

[0012] S5. Dissolve chitosan and the hydroxyapatite obtained in S3 uniformly by ultrasonic treatment with acetic acid, add acrylic acid-modified β-cyclodextrin and the peptide-calcium chelate, treat at 60 - 70 °C for 60 - 90 min, and then add fish scale collagen and treat at 30 - 45 °C for 3 - 6 h to prepare a fish scale composite hydrogel.

[0013] Furthermore, in the step S1, the mass ratio of fish scale powder to water is 1:(10 - 15).

[0014] Furthermore, in the step S2, the specific operation of the high-frequency - low-frequency acoustic wave treatment is that the high-frequency acoustic wave treatment and the low-frequency acoustic wave treatment are alternately carried out 3 - 6 times; the conditions of the high-frequency acoustic wave treatment are that the acoustic wave frequency is 0.5 - 1 MHz and the treatment time is 10 - 20 min; the conditions of the low-frequency acoustic wave treatment are that the acoustic wave frequency is 20 - 50 kHz and the treatment time is 40 - 60 min.

[0015] Furthermore, in the step S3, the mass ratio of the fish scale filtrate to water is 1:(20 - 30); the hot pressing treatment temperature is 110 - 130 °C and the treatment time is 30 - 60 min.

[0016] Furthermore, in the step S3, the conditions of enzymatic hydrolysis are that the enzyme addition amount is 2 - 6 wt%, the enzymatic hydrolysis temperature is 50 - 55 °C, and the enzymatic hydrolysis time is 3 - 5 h; the protease used for enzymatic hydrolysis is papain, neutral protease or alkaline protease.

[0017] Furthermore, in the step S3, the ionic liquid is a mixed solution of choline chloride and 1,4-butanediol; the conditions for ionic liquid extraction are that the extraction temperature is 50 - 60 °C, the extraction time is 1.5 - 2.5 h, and the material-liquid ratio of the residue to the ionic liquid is 1:(10 - 15).

[0018] Furthermore, in the step S5, the mass ratio of chitosan, hydroxyapatite and acrylic acid-modified β-cyclodextrin is (0.5 - 1.5):(1 - 3):(1.5 - 4).

[0019] Furthermore, in the step S5, the addition amount of the peptide-calcium chelate is 2.5 - 5 wt%; the addition amount of the fish scale collagen is 2 - 10 wt%.

[0020] The fish scale composite hydrogel prepared by the method described in any one of the above.

[0021] Furthermore, the application of the fish scale composite hydrogel in drug delivery.

[0022] Beneficial effects

[0023] 1. In the preparation of the fish scale composite hydrogel of the present invention, high-frequency and low-frequency acoustic waves are alternately used. On the one hand, when high-frequency acoustic waves are applied to the fish scale powder solution, cavitation will cause cavitation effect and generate microbubble nuclei through cavitation. The microbubble nuclei rapidly aggregate and grow energy through oscillation, growth, contraction, and collapse, and release huge energy when rupturing. At the same time, the intense mechanical vibration generated by the high-frequency acoustic waves synergistically destroys the plywood structure between collagen fibers and hydroxyapatite in the fish scales, promoting the dissociation of collagen from the bondage of the apatite lattice. On the other hand, low-frequency acoustic waves can further destroy the binding force between collagen and hydroxyapatite, help loosen the connection between collagen and hydroxyapatite, promote the dissociation of collagen while having less impact on the structure of collagen, thus retaining the biological activity and functionality of collagen.

[0024] 2. The fish scale hydrogel prepared by the present invention has a unique "jellyfish" structure. On the one hand, the gel structure nuclear layer acting as the umbrella-shaped head of the jellyfish is composed of β-cyclodextrin inclusion of hydroxyapatite and chitosan. Chitosan and hydroxyapatite form a cross-linked network structure through ionic coordination bonds and hydrogen bond interactions under the cross-linking action of peptide calcium chelate, and are included in the internal cavity structure of β-cyclodextrin to form the fish scale hydrogel structure nuclear layer. On the other hand, the acrylic groups of acrylic acid-modified β-cyclodextrin form covalently cross-linked long chains with the amino groups in collagen and attach to the outer part of the cyclic cavity of β-cyclodextrin to form the branched tentacle structure of the fish scale hydrogel.

[0025] 3. Both the jellyfish structure gel nuclear layer and the branched tentacle structure in the fish scale hydrogel prepared by the present invention can be applied to drug loading. The drug carried by the gel at the branched tentacle structure is rapidly released and can be used for the acute treatment of diseases, alleviating the adverse reactions caused by the diseases first. At the same time, the drug carried by the gel in the gel nuclear layer is slowly released and can be used for the later treatment of diseases to achieve gradient drug release control. In addition, the branched tentacle structure of the fish scale hydrogel can make the hydrogel tightly adhere to the drug release site, which is beneficial to the precise release and exertion of the drug.

[0026] 4. The method for treating fish scales of the present invention can solve the problem of collagen denaturation caused by traditional acid-base treatment of fish scales, ensure the biological value of collagen and hydroxyapatite extracted from fish scales, and at the same time, the preparation of peptide calcium chelate can improve the utilization rate of fish scale raw materials; in addition, the preparation method of fish scale hydrogel can provide good technical support for the high-value utilization of fish scales. Brief description of the drawings

[0027] Figure 1 For the collagen extraction rate, peptide calcium chelation rate, and hydroxyapatite extraction rate of Examples 1-5 and Comparative Examples 1-2;

[0028] Figure 2Drug loading of Examples 6 - 12 and Comparative Examples 3 - 7;

[0029] Figure 3 Drug cumulative release rate of Example 9 and Comparative Examples 3 - 7;

[0030] Figure 4 Cell viability of Example 9 and Comparative Examples 3 - 7. Detailed implementation manners

[0031] The present invention will be further described below in conjunction with examples. The following examples are explanations of the present invention and the present invention is not limited to the following examples:

[0032] Example 1

[0033] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0034] S1. The fish scales are first cleaned to remove impurities, dried and pulverized, and passed through an 80 - mesh sieve to obtain fish scale powder. 100 g of fish scale powder is added to 1000 g of water to prepare a fish scale powder solution;

[0035] S2. The fish scale powder solution is treated by high - frequency - low - frequency acoustic waves in a cycle of 4 times. The acoustic wave frequency for high - frequency acoustic wave treatment is 0.5 MHz and the treatment time is 10 min. The conditions for low - frequency acoustic wave treatment are acoustic wave frequency 30 kHz and treatment time 45 min; then it is filtered to obtain fish scale filtrate and calcium - ion - containing filtrate;

[0036] S3. 50 g of fish scale filtrate is added to 1250 g of water, and hot - pressed at 110 °C for 40 min, then filtered, concentrated and dried to obtain fish scale collagen; the remaining residue is added to 15 times the volume of choline chloride + 1,4 - butanediol solution, treated at 55 °C for 2 h, and then filtered, washed and purified to obtain hydroxyapatite;

[0037] S4. 10 g of fish scale collagen is dissolved evenly in 100 g of water, and then 2 g of papain is added and enzymolyzed at 50 °C for 4 h to obtain fish scale collagen peptide; the fish scale collagen peptide is mixed evenly with the calcium - ion - containing filtrate in S2, treated at pH 8.0 and 50 °C for 30 min, and then centrifuged, washed and dried to prepare a peptide - calcium chelate.

[0038] Example 2

[0039] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0040] S1. The fish scales are first cleaned to remove impurities, dried and pulverized, and passed through an 80 - mesh sieve to obtain fish scale powder. 100 g of fish scale powder is added to 1500 g of water to prepare a fish scale powder solution;

[0041] S2. Treat the fish scale powder solution with high-frequency and low-frequency sound waves in a cycle of 4 times. The sound wave frequency for high-frequency sound wave treatment is 0.5 MHz and the treatment time is 10 min. The conditions for low-frequency sound wave treatment are a sound wave frequency of 30 kHz and a treatment time of 45 min; then filter to obtain fish scale filtrate and calcium ion-containing filtrate;

[0042] S3. Add 50 g of fish scale filtrate to 1250 g of water, and conduct hot pressing treatment at 110 °C for 30 min, then filter, concentrate, and dry to obtain fish scale collagen; add the remaining residue to 15 times the volume of choline chloride + 1,4-butanediol solution, and treat it at 55 °C for 1.5 h, then filter, wash, and purify to obtain hydroxyapatite;

[0043] S4. Dissolve 10 g of fish scale collagen evenly in 100 g of water, then add 3 g of papain and enzymatically hydrolyze at 50 °C for 3 h to obtain fish scale collagen peptides; mix the fish scale collagen peptides evenly with the calcium ion-containing filtrate in S2, and treat it at pH 8.0 and 50 °C for 30 min, then centrifuge, wash, and dry to make a peptide-calcium chelate.

[0044] Example 3

[0045] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0046] S1. First, clean, remove impurities, dry, pulverize the fish scales, and pass through an 80-mesh sieve to obtain fish scale powder. Prepare a fish scale powder solution by adding 100 g of fish scale powder to 1000 g of water;

[0047] S2. Treat the fish scale powder solution with high-frequency and low-frequency sound waves in a cycle of 4 times. The sound wave frequency for high-frequency sound wave treatment is 0.75 MHz and the treatment time is 10 min. The conditions for low-frequency sound wave treatment are a sound wave frequency of 30 kHz and a treatment time of 45 min; then filter to obtain fish scale filtrate and calcium ion-containing filtrate;

[0048] S3. Add 50 g of fish scale filtrate to 1250 g of water, and conduct hot pressing treatment at 110 °C for 40 min, then filter, concentrate, and dry to obtain fish scale collagen; add the remaining residue to 15 times the volume of choline chloride + 1,4-butanediol solution, and treat it at 55 °C for 2 h, then filter, wash, and purify to obtain hydroxyapatite;

[0049] S4. Dissolve 10 g of fish scale collagen evenly in 100 g of water, then add 2 g of papain and enzymatically hydrolyze at 50 °C for 4 h to obtain fish scale collagen peptides; mix the fish scale collagen peptides evenly with the calcium ion-containing filtrate in S2, and treat it at pH 8.0 and 50 °C for 45 min, then centrifuge, wash, and dry to make a peptide-calcium chelate.

[0050] Example 4

[0051] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0052] S1. The fish scales are first cleaned to remove impurities, dried and crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100 g of fish scale powder is added to 1000 g of water to prepare a fish scale powder solution;

[0053] S2. The fish scale powder solution is treated by high-frequency - low-frequency acoustic waves in a cycle of 4 times. Among them, the acoustic wave frequency of the high-frequency acoustic wave treatment is 0.5 MHz and the treatment time is 10 min, and the treatment conditions of the low-frequency acoustic wave are an acoustic wave frequency of 50 kHz and a treatment time of 45 min; then it is filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate;

[0054] S3. 50 g of the fish scale filtrate is added to 1250 g of water, and hot-pressed at 110 °C for 40 min, then filtered, concentrated and dried to obtain fish scale collagen; the remaining residue is added with 12.5 times the volume of choline chloride + 1,4-butanediol solution, treated at 60 °C for 2 h, and filtered, washed and purified to obtain hydroxyapatite;

[0055] S4. 10 g of fish scale collagen is dissolved evenly in 100 g of water, and then 4 g of papain is added and enzymatically hydrolyzed at 50 °C for 4 h to obtain fish scale collagen peptides; the fish scale collagen peptides are mixed evenly with the calcium ion-containing filtrate in S2, treated at pH 8.0 and 55 °C for 30 min, and centrifuged, washed and dried to prepare a peptide-calcium chelate.

[0056] Example 5

[0057] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0058] S1. The fish scales are first cleaned to remove impurities, dried and crushed, and passed through an 80-mesh sieve to obtain fish scale powder. 100 g of fish scale powder is added to 1000 g of water to prepare a fish scale powder solution;

[0059] S2. The fish scale powder solution is treated by high-frequency - low-frequency acoustic waves in a cycle of 4 times. Among them, the acoustic wave frequency of the high-frequency acoustic wave treatment is 0.5 MHz and the treatment time is 10 min, and the treatment conditions of the low-frequency acoustic wave are an acoustic wave frequency of 30 kHz and a treatment time of 45 min; then it is filtered to obtain a fish scale filtrate and a calcium ion-containing filtrate;

[0060] S3. 50 g of the fish scale filtrate is added to 1250 g of water, and hot-pressed at 120 °C for 40 min, then filtered, concentrated and dried to obtain fish scale collagen; the remaining residue is added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55 °C for 2 h, and filtered, washed and purified to obtain hydroxyapatite;

[0061] Dissolve 10 g of fish scale collagen in 100 g of water evenly, then add 2 g of papain and enzymatically hydrolyze at 50 °C for 4 h to obtain fish scale collagen peptide; mix the fish scale collagen peptide evenly with the calcium ion-containing filtrate in S2, and treat it at pH 8.0 and 50 °C for 30 min, then centrifuge, wash and dry to make a peptide-calcium chelate.

[0062] Comparative Example 1

[0063] The difference between this comparative example and Example 3 is that high-frequency sound wave treatment is not used.

[0064] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0065] S1. First, clean, remove impurities, dry, crush and sieve the fish scales through an 80-mesh sieve to obtain fish scale powder, and prepare a fish scale powder solution by adding 1000 g of water to 100 g of fish scale powder;

[0066] S2. Use low-frequency sound waves to circulate and treat the fish scale powder solution 4 times, where the low-frequency sound wave treatment conditions are a sound wave frequency of 30 kHz and a treatment time of 45 min; then filter to obtain a fish scale filtrate and a calcium ion-containing filtrate;

[0067] S3. Add 50 g of the fish scale filtrate to 1250 g of water, and perform hot pressing treatment at 110 °C for 40 min, then filter, concentrate and dry to obtain fish scale collagen; add 15 times the volume of choline chloride + 1,4-butanediol solution to the remaining residue, and treat it at 55 °C for 2 h, then filter, wash and purify to obtain hydroxyapatite;

[0068] Dissolve 10 g of fish scale collagen in 100 g of water evenly, then add 2 g of papain and enzymatically hydrolyze at 50 °C for 4 h to obtain fish scale collagen peptide; mix the fish scale collagen peptide evenly with the calcium ion-containing filtrate in S2, and treat it at pH 8.0 and 50 °C for 45 min, then centrifuge, wash and dry to make a peptide-calcium chelate.

[0069] Comparative Example 2

[0070] The difference between this comparative example and Example 3 is that high-frequency-low-frequency sound wave treatment is not used.

[0071] A method for extracting fish scale collagen and hydroxyapatite from fish scales, comprising the following steps:

[0072] S1. First, clean, remove impurities, dry, crush and sieve the fish scales through an 80-mesh sieve to obtain fish scale powder, and prepare a fish scale powder solution by adding 1000 g of water to 100 g of fish scale powder;

[0073] S2. The fish scale powder solution is subjected to hot pressing treatment at 110 °C for 40 min, filtered, concentrated and dried to obtain fish scale collagen; the remaining residue is added with 15 times the volume of choline chloride + 1,4-butanediol solution, treated at 55 °C for 2 h, filtered, washed and purified to obtain hydroxyapatite;

[0074] S3. 10 g of fish scale collagen is dissolved evenly in 100 g of water, and then 2 g of papain is added and enzymolyzed at 50 °C for 4 h to obtain fish scale collagen peptide; the fish scale collagen peptide is mixed evenly with the filtrate in S2, treated at pH 8.0 and 50 °C for 45 min, centrifuged, washed and dried to prepare a peptide-calcium chelate.

[0075] Index determination

[0076] (1)Collagen extraction rate

[0077] Analyze the collagen extraction rates of Examples 1-5, Comparative Example 1 and Comparative Example 2 according to the hydroxyproline content determination method in GB / T9695.23-2008.

[0078] (2)Hydroxyapatite extraction rate

[0079] Weigh the weight of the residue (denoted as M1) and the weight of hydroxyapatite (denoted as M2) respectively, and calculate the hydroxyapatite extraction rate through the following formula.

[0080] Hydroxyapatite extraction rate (%) = M1 / M2 × 100%

[0081] (3)Peptide-calcium chelation rate

[0082] Determine the calcium content of Examples 1-5, Comparative Example 1 and Comparative Example 2 according to the calcium content determination method in GB 5009.92-2016, and calculate the peptide-calcium chelation rate through the following formula.

[0083] Peptide-calcium chelation rate (%) = calcium content in peptide-calcium chelate / calcium content in filtrate × 100%

[0084] From Figure 1It can be seen that the collagen extraction rate, peptide-calcium chelation rate, and hydroxyapatite extraction rate in Examples 1-5 are all higher than those in Comparative Example 1 and Comparative Example 2. In Comparative Example 1, only low-frequency acoustic waves were used for treatment. Since the destructive effect of low-frequency acoustic waves on fish scales during the pretreatment stage is smaller than that of high-frequency acoustic waves, the plywood structure between collagen and hydroxyapatite in fish scales cannot be fully destroyed, resulting in incomplete dissociation of collagen from hydroxyapatite, thus reducing the collagen extraction rate and hydroxyapatite extraction rate. In addition, in Comparative Example 2, high-frequency-low-frequency acoustic wave treatment was not used, and it was even less able to effectively damage the plywood structure between collagen and hydroxyapatite in fish scales during the fish scale pretreatment step. Collagen could not be dissociated, which affected the subsequent extraction of hydroxyapatite, so the collagen extraction rate and hydroxyapatite extraction rate were reduced. Therefore, the following preparation method of the fish scale composite hydrogel uses the fish scale collagen, hydroxyapatite, and peptide-calcium chelate extracted in Example 3.

[0085] Example 6

[0086] A preparation method of a fish scale composite hydrogel includes the following steps:

[0087] Step 1. Dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until evenly dissolved;

[0088] Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide-calcium chelate and treat at 60 °C for 60 min;

[0089] Step 3. Add 5 g of fish scale collagen and treat at 40 °C for 6 h to obtain the fish scale composite hydrogel.

[0090] Example 7

[0091] A preparation method of a fish scale composite hydrogel includes the following steps:

[0092] Step 1. Dissolve 5 g of chitosan and 20 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until evenly dissolved;

[0093] Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide-calcium chelate and treat at 60 °C for 60 min;

[0094] Step 3. Add 5 g of fish scale collagen and treat at 30 °C for 4 h to obtain the fish scale composite hydrogel.

[0095] Example 8

[0096] A preparation method of a fish scale composite hydrogel includes the following steps:

[0097] Step 1. Dissolve 10 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until evenly dissolved;

[0098] Step 2. Add 20 g of acrylate-modified β-cyclodextrin and 3 g of peptide calcium chelate and process at 60 °C for 60 min;

[0099] Step 3. Add 5 g of fish scale collagen and process at 40 °C for 6 h to obtain a fish scale composite hydrogel.

[0100] Example 9

[0101] A method for preparing a fish scale composite hydrogel, comprising the following steps:

[0102] Step 1. Ultrasonically dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution until homogeneous;

[0103] Step 2. Add 30 g of acrylate-modified β-cyclodextrin and 3 g of peptide calcium chelate and process at 60 °C for 60 min;

[0104] Step 3. Add 5 g of fish scale collagen and process at 30 °C for 4 h to obtain a fish scale composite hydrogel.

[0105] Example 10

[0106] A method for preparing a fish scale composite hydrogel, comprising the following steps:

[0107] Step 1. Ultrasonically dissolve 5 g of chitosan and 5 g of hydroxyapatite in 100 mL of acetic acid solution until homogeneous;

[0108] Step 2. Add 30 g of acrylate-modified β-cyclodextrin and 3 g of peptide calcium chelate and process at 60 °C for 60 min;

[0109] Step 3. Add 5 g of fish scale collagen and process at 40 °C for 6 h to obtain a fish scale composite hydrogel.

[0110] Example 11

[0111] A method for preparing a fish scale composite hydrogel, comprising the following steps:

[0112] Step 1. Ultrasonically dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution until homogeneous;

[0113] Step 2. Add 20 g of acrylate-modified β-cyclodextrin and 2.5 g of peptide calcium chelate and process at 60 °C for 60 min;

[0114] Step 3. Add 5 g of fish scale collagen and process at 40 °C for 6 h to obtain a fish scale composite hydrogel.

[0115] Example 12

[0116] A method for preparing a fish scale composite hydrogel, comprising the following steps:

[0117] Step 1. Dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until evenly dissolved;

[0118] Step 2. Add 20 g of acrylic acid modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60 °C for 60 min;

[0119] Step 3. Add 10 g of fish scale collagen and treat at 40 °C for 6 h to obtain the fish scale composite hydrogel.

[0120] Comparative Example 3

[0121] The difference between this comparative example and Example 6 is that the peptide calcium chelate is replaced with genipin.

[0122] A preparation method of a fish scale composite hydrogel, comprising the following steps:

[0123] Step 1. Dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until evenly dissolved;

[0124] Step 2. Add 20 g of acrylic acid modified β-cyclodextrin and 3 g of genipin and treat at 60 °C for 60 min;

[0125] Step 3. Add 5 g of fish scale collagen and treat at 40 °C for 6 h to obtain the fish scale composite hydrogel.

[0126] Comparative Example 4

[0127] The difference between this comparative example and Example 6 is that the peptide calcium chelate is replaced with glutaraldehyde.

[0128] A preparation method of a fish scale composite hydrogel, comprising the following steps:

[0129] Step 1. Dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until evenly dissolved;

[0130] Step 2. Add 20 g of acrylic acid modified β-cyclodextrin and 3 g of glutaraldehyde and treat at 60 °C for 60 min;

[0131] Step 3. Add 5 g of fish scale collagen and treat at 40 °C for 6 h to obtain the fish scale composite hydrogel.

[0132] Comparative Example 5

[0133] The difference between this comparative example and Example 6 is that no peptide calcium chelate is added.

[0134] A preparation method of a fish scale composite hydrogel, comprising the following steps:

[0135] Step 1. Dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until homogeneous.

[0136] Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and treat at 60 °C for 60 min.

[0137] Step 3. Add 5 g of fish scale collagen and treat at 40 °C for 6 h to obtain a fish scale composite hydrogel.

[0138] Comparative Example 6

[0139] The difference between this comparative example and Example 6 is that hydroxyapatite is not added.

[0140] A preparation method of a fish scale composite hydrogel, comprising the following steps:

[0141] Step 1. Dissolve 5 g of chitosan in 100 mL of acetic acid solution by ultrasonic treatment until homogeneous.

[0142] Step 2. Add 20 g of acrylic acid-modified β-cyclodextrin and 3 g of peptide calcium chelate and treat at 60 °C for 60 min.

[0143] Step 3. Add 5 g of fish scale collagen and treat at 40 °C for 6 h to obtain a fish scale composite hydrogel.

[0144] Comparative Example 7

[0145] The difference between this comparative example and Example 6 is that acrylic acid-modified β-cyclodextrin is not added.

[0146] A preparation method of a fish scale composite hydrogel, comprising the following steps:

[0147] Step 1. Dissolve 5 g of chitosan and 10 g of hydroxyapatite in 100 mL of acetic acid solution by ultrasonic treatment until homogeneous.

[0148] Step 2. Add 3 g of peptide calcium chelate and treat at 60 °C for 60 min.

[0149] Step 3. Add 5 g of fish scale collagen and treat at 40 °C for 6 h to obtain a fish scale composite hydrogel.

[0150] Performance measurement

[0151] (1) Drug loading

[0152] Using curcumin as a model drug, weigh 50 mg of the fish scale composite hydrogel, place it in 10 mL of an ethanol solution of curcumin with a mass concentration of 1.0 mg / mL, mix evenly for 24 h, and then at 7.5×10 3Centrifuge at

[0153] It can be seen from Figure 2 that the drug loading amounts of Examples 6-12 are all higher than those of Comparative Examples 3-7. Among them, the drug loading amount of Example 9 is the highest, indicating that the core layer of the fish scale hydrogel structure is prepared by using acrylic acid-modified β-cyclodextrin, chitosan, hydroxyapatite and peptide calcium chelate. Chitosan and peptide calcium chelate act through ionic coordination bonds and hydrogen bonds. At the same time, hydroxyapatite is filled into the cross-linked network to support the stability of the network and is included in the internal cavity structure of β-cyclodextrin to form the core layer of the fish scale hydrogel structure. In addition, the acrylic acid group forms a covalently cross-linked long chain with the amino group in collagen and attaches to the outside of the cyclic cavity of β-cyclodextrin to form the branched whisker structure of the fish scale hydrogel. The two can encapsulate curcumin to the greatest extent, increase the loading amount of curcumin, and thus increase the drug loading amount of the fish scale composite hydrogel.

[0154] In Comparative Example 3 and Comparative Example 4, genipin and glutaraldehyde are used for cross-linking respectively, and their cross-linking effects with chitosan and hydroxyapatite are weaker than those of peptide calcium chelate. In Comparative Example 5 and Comparative Example 6, peptide calcium chelate and hydroxyapatite are not added respectively, which will affect the stability of the core layer structure of the fish scale composite hydrogel. In addition, in Comparative Example 7, acrylic acid-modified β-cyclodextrin is not used, and the prepared fish scale composite hydrogel does not form a branched whisker structure. Therefore, the drug loading amount of the fish scale composite hydrogel will be reduced.

[0155] (2) Cumulative drug release rate

[0156] Carry out an in vitro release experiment on the curcumin-loaded fish scale composite hydrogel according to the regulations of the 2015 edition of the Chinese Pharmacopoeia. The specific method is as follows: Use a dilute hydrochloric acid solution (pH = 1.4) with a mass fraction of 20% ethanol as a simulated artificial gastric juice for in vitro drug release testing. At the same time, prepare a cut-off relative molecular mass of 8×10 3 -10×10 3For the dialysis bag, weigh 50 mg of the drug-loaded sample and place it therein, then put the dialysis bag into the sustained-release solution, stir it under the environmental condition of a 37.5 °C water bath, start the in vitro release test of curcumin. Take out 3 mL of the sustained-release solution at the preset time points, and immediately add 3 mL of the corresponding sustained-release solution when the sustained-release solution is taken out. Use an ultraviolet spectrophotometer to measure the absorbance of the sample solution at 427 nm, and calculate the drug cumulative release rate according to the following formula;

[0157] Drug cumulative release rate (%) = (V0c n +V ) / m × 100%, where V0 is the total volume of the solution; V is the volume of the solution taken out each time; i = 1, 2......n is the number of times of sucking the solution; ci is the drug concentration measured when the i-th solution is obtained; m is the total mass of curcumin encapsulated in the drug-loaded hydrogel used for testing.

[0158] It can be seen from Figure 3 that the drug cumulative release rate of curcumin carried by the fish scale composite hydrogel prepared in Example 9 is lower than that of Comparative Examples 3 - 7 during long-term action, and the curcumin loaded by the branched whisker structure of the fish scale composite hydrogel has a burst release in a short time, which can achieve the maximum degree of remission at the initial stage when actually treating diseases with drugs; in addition, the curcumin on the surface of the synthetic structure of the fish scale composite hydrogel needs to be released from the network of chitosan-hydroxyapatite first, and then enter the gastric juice through the pores on the surface of the β-cyclodextrin inclusion structure, which is equivalent to constructing a double-layer controlled release structure to extend the sustained release time of the drug, so as to achieve the effect of gradient controlled release of curcumin.

[0159] (3) Biocompatibility

[0160] Refer to the biological testing rules of ISO10993.1-2018 for medical devices to carry out in vitro cytotoxicity experiments on the medium raw materials, and use the CCK-8 method to detect cytotoxicity. Weigh 0.9 g of the sample, soak it in industrial ethanol for 24 h, filter and dry it, and then sterilize it with ultraviolet light; soak the sample in 20 mL of complete medium, incubate it at 37 °C for 24 h, filter it with a microporous membrane, and recover the leachate, using the complete culture medium as a control experiment; for in vitro cell experiments, inject Chinese hamster uterine cells into a 96-well plate, with 10 wells in each group; according to the classification method, add 200 μL of the leachate or complete culture medium (control) of different samples to each group. After culturing for 24 h, 48 h, and 72 h, add CCK-8 assay reagent to each well, gently shake it, and then put it into a constant temperature cell incubator for further culture. After 2 - 3 h, use an instant enzyme-linked immunosorbent assay instrument to measure the absorbance of each well sample at a wavelength of 450 nm, and calculate the relative cell proliferation rate.

[0161] It can be seen from Figure 4It can be seen that with the extension of the culture time, the relative cell proliferation rates of Example 9 are all greater than 90%, indicating that the scale composite hydrogel prepared in Example 9 has almost no toxicity to cell proliferation. In Comparative Example 3 and Comparative Example 4, genipin and glutaraldehyde were used to crosslink and prepare the scale composite hydrogel, respectively. Both genipin and glutaraldehyde have certain cytotoxicity, and the cytotoxicity of glutaraldehyde is stronger, which inhibits cell proliferation and reduces the relative cell proliferation rate during the cell culture and proliferation process; Comparative Examples 5-7 have little effect on the relative cell proliferation rate.

[0162] As mentioned above, the above are only the preferred embodiments of the present invention, and there is no any formal limitation to the present invention. Any person skilled in the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for preparing a fish scale composite hydrogel, characterized in that: The following steps are involved: S1. The fish scales are first cleaned, dried, crushed, and sieved to obtain fish scale powder, and water is added to prepare a fish scale powder solution; S2. The fish scale powder solution is treated with high-frequency-low-frequency sonic waves to obtain a fish scale filtrate and a calcium ion-containing filtrate by filtering; the specific operation of the high-frequency-low-frequency sonic wave treatment is that the high-frequency sonic wave treatment and the low-frequency sonic wave treatment are alternately performed 3-6 times; the high-frequency sonic wave treatment condition is a sonic wave frequency of 0.5-1MHz and a treatment time of 10-20min; the low-frequency sonic wave treatment condition is a sonic wave frequency of 20-50kHz and a treatment time of 40-60min; S3. The fish scale filtrate is added with water and then subjected to hot pressing to obtain fish scale collagen and residue, wherein the residue is subjected to ionic liquid extraction, dried to obtain hydroxyapatite, and a portion of the fish scale collagen is enzymatically hydrolyzed to obtain fish scale collagen peptides; S4. treating the fish scale collagen peptide and the calcium ion-containing filtrate obtained in S2 at pH 6.5-8.0 and 45-55° C. for 30-45 min to obtain a peptide calcium chelate; S5. Add acetic acid to chitosan and hydroxyapatite obtained in S3 and ultrasonically dissolve them uniformly, add acrylic acid-modified β-cyclodextrin and peptide calcium chelate and treat them at 60-70°C for 60-90 minutes, then add fish scale collagen and treat them at 30-45°C for 3-6 hours to obtain fish scale composite hydrogel.

2. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The mass ratio of the fish scale powder to water in step S1 is 1:(10-15).

3. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: In step S3, the mass ratio of the fish scale filtrate to water is 1:(20-30); the hot pressing treatment temperature is 110-130° C. and the treatment time is 30-60 min.

4. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The conditions for enzymolysis in step S3 are: an enzyme addition amount of 2-6wt%, an enzymolysis temperature of 50-55°C, and an enzymolysis time of 3-5h; and the protease used in the enzymolysis is papain, neutral protease or alkaline protease.

5. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: The ionic liquid in step S3 is a mixed solution of choline chloride and 1,4-butanediol; the conditions for the ionic liquid extraction are extraction temperature of 50-60°C, extraction time of 1.5-2.5h, and a solid-liquid ratio of residue to ionic liquid of 1:(10-15).

6. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: In the step S5, the mass ratio of chitosan, hydroxyapatite and acrylic acid-modified β-cyclodextrin is (0.5-1.5):(1-3):(1.5-4).

7. The method for preparing a fish scale composite hydrogel according to claim 1, characterized in that: In the step S5, the amount of peptide calcium chelate added is 2.5-5wt%; the amount of fish scale collagen added is 2-10wt%.

8. The fish scale composite hydrogel prepared according to the method according to any one of claims 1 to 7.

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